**4. Discussion**

Recently, it was revealed that TRP channels are expressed throughout the human body, including in immune cells, sensory nerves, and resident tissue cells [4–7]. Saunders et al. examined the expression of TRPV1 and TRPV2 in PBMCs from healthy subjects and speculated that as they have a role in the detection of noxious stimuli in the blood or under pathological conditions, their upregulation acts as an indicator of inflammation [13,21]. However, to the best of our knowledge, the current study is the first to explore the relationships among the gene expression profiles of TRP channel members in PBMCs from patients with IBD.

TRPV1, a polymodal receptor involved in inflammation and nociception, plays an important role in visceral hypersensitivity [22]. Reportedly, TRPV1-immunoreactivity in colonic tissue is increased, and is correlated with the severity of abdominal pain in patients with IBD [22,23]. More recently, TRPV1 was shown to be expressed in CD4+ T cells, where it regulates cell activation and proinflammatory properties in IBD [24]. Meanwhile, a TRPV1 antagonist suppressed colitis and colorectal distension in animal models [25]. In a study, two-fold upregulation of the TRPV1 gene was found in PBMCs of patients hyposensitive to capsaicin, pain, and thermal stimuli [14]. However, in the present study, we did not observe any significant differences in TRPV1 expression in PBMCs from patients with IBD compared to healthy controls. Hence, further studies are needed to determine the role of the TRPV1 pathway in PBMCs from IBD patients using protein or functional data.

TRPV2 triggers a wide range of physiological actions including changes in the innate and adaptive immune system. It is expressed in granulocytes and monocytes/macrophages and contributes to phagocytosis, migration, and inflammatory cytokine production [26]. In PBMCs, the increased expression of TRPV2 was closely correlated with childhood asthma [15]. In TRPV2-knockout mice, colitis was less severe due to the reduced infiltration of macrophages [27], suggesting that the TRPV2 pathway plays a key role in the development of colitis. Our study showed that TRPV2 expression in PBMCs decreased in patients with UC and CD and was inversely correlated with disease activity. Based on these findings, a reduction in TRPV2 expression in PBMCs may hypothetically dampen the proinflammatory response and could possibly reduce the severity of intestinal inflammation. However, further investigations are required to confirm this hypothesis.

TRPV3 is broadly expressed in intestinal epithelial cells, possibly for nutrient sensing and digestion [28,29]; however, its precise function is not well understood. With the exception of one study reporting its association with a higher risk of colorectal cancer, none others have investigated the role of TRPV3 in gu<sup>t</sup> disease [30]. Interestingly, a recent study demonstrated a decrease in the proliferation rate of oral epithelial cells in TRPV3-knockout mice [31], suggesting that TRPV3 may contribute to oral

wound repair. Our data revealed that TRPV3 expression was only decreased in PBMCs of patients with CD, however, its function in PBMCs requires further investigation.

TRPV4 is expressed and is functional in intestinal epithelial cells, glial cells, and CD45+ leukocytes; its activation in the gu<sup>t</sup> causes increased intracellular calcium concentrations and chemokine release [28]. Studies have indicated a strong role for TRPV4 in IBD with elevated expression of TRPV4 observed in the intestinal tissue of patients with UC and CD [32]. In animal models of colitis, TRPV4 activation causes inflammation [32], and its blockade alleviates inflammation [33]. In this study, we found that the expression of TRPV4 was also upregulated in PBMCs and was correlated with the CRP level in patients with CD. Although the correlations were quite weak, and conclusions cannot be drawn based on mRNA levels without protein and modulation data, our data suggests a potential role for leukocyte TRPV4 in the pathophysiology of CD.

Recent studies have also revealed the involvement of TRPM2 in various aspects of immunity [34]. In a colitis model, TRPM2 has been implicated in inflammatory pathways, specifically as a key participant in chemokine production [35]. Contrary to this proinflammatory action, TRPM2-knockout mice exhibited decreased survival after liver infection with *Listeria monocytogenes* [36]. Thus, TRPM2 may be detrimental or beneficial depending on the underlying disease. Although no information is available on TRPM2 expression in the intestinal tissue, its upregulation in PBMCs of patients with UC and CD is of particular interest as a participant in disease pathogenesis and as a promising marker for disease activity.

TRPM4 plays a predominant role as a negative feedback mechanism during calcium oscillations, which may be important for di fferential gene expression in T cells [37]. A recent study showed that TRPM4 plays an important role in the immune surveillance processes. It is essential for the proper functioning of monocytes/macrophages and the e fficiency of the subsequent response to infection [38], as well as the migration of dendritic cells [39]. At present, no information is available on the role of TRPM4 in gu<sup>t</sup> disease. Our study showed no alteration in TRPM4 expression in PBMCs from either UC or CD patients, however, further investigation is needed.

Knowledge of the role of TRPM5, predominantly expressed by tuft cells that are an intestinal epithelial subset [40], in gu<sup>t</sup> disease is limited [41]. A recent report showed that the disruption of chemosensory signaling through the loss of TRPM5 abrogates the expansion of tuft cells [42]. Interestingly, the ablation of doublecortin-like kinase 1 (DCLK1), a marker of tuft cells, in the colonic epithelium exacerbates colitis in mice [43,44]. This finding suggests that TRPM5 plays an important role in regulating the intestinal inflammatory response and epithelial integrity. Another study showed that the number of DCLK1-positive cells decreased in intestinal tissue from patients with celiac disease [45]. Meanwhile, our study found no alteration in TRPM5 expression in PBMCs from either UC or CD patients, warranting further investigation.

The TRPC subfamily comprises six members (TRPC1, TRPC3–7) in humans, many of which are ubiquitously expressed in tissues and modulate a multitude of cellular responses [46]. Of these, TRPC1, TRPC3, and TRPC6 were detectable in this study. As TRPC1 controls the release of interleukin-1 from macrophages [47] and that of tumor necrosis factorα from mast cells [48], its decreased expression in PBMCs from patients with UC and CD seen in our study may enhance disease development. Moreover, the expression of TRPC6 was upregulated in PBMCs from patients with CD but not those with UC in this study. These results, together with those of a previous study reporting increased TRPC6 mRNA levels in stenotic areas of patients with CD [49], sugges<sup>t</sup> that TRPC6 may be associated with excessive CD fibrosis.

To date, the mechanisms of TRP channel expression in PBMCs from patients with IBD remain elusive. TRP channels in PBMCs may respond to multiple stimuli present in the peripheral circulation of these patients. The di fferent expression levels of these TRP channels may sugges<sup>t</sup> a possible role as an indicator of inflammation at secondary sites, as well as involvement in IBD pathophysiology. Additionally, the di fference in expression of TRPV channels in UC and CD patients may result from di fferences in the disease pathogenesis. Recent studies have also shown that lipopolysaccharide (LPS) activates several members of the TRP channel family, such as TRPV4 and TRPA1, as well as the Toll-like receptor 4 (TLR4), suggesting the role of TRP channels as sensors of bacterial endotoxins, and therefore, as crucial players in innate immunity. Moreover, since TRP channel and TLR expression overlap in many cell types, including immune cells and epithelial cells, it would be of interest to explore the crosstalk between intracellular signaling pathways initiated by TLR activation and TRP channel activation in patients with IBD [50]. Understanding the involvement of TRP channel members in IBD will be crucial to evaluate the potential for manipulating TRP activity as a therapeutic intervention [51,52].

As the majority of our patients were receiving medications, we assessed whether medical treatment may a ffect the expression of TRP channel family in PBMCs. A subgroup analysis among patients with untreated and treated IBD showed that the use of medications had no significant e ffect on mRNA levels of any TRP. Future studies sequentially assessing mRNA levels of TRPs in the same patient are required to confirm this lack of association.

This study had certain limitations. First, it was conducted at a single center and involved a limited number of patients, which could cause a β-error, particularly for the analysis of the medical treatment. Second, as this study analyzed the gene expression of all PBMCs, the PBMC subsets that actually express TRP channels remain to be determined. Third, this study characterized the TRP expression at the mRNA level only. To support any conclusion on the role of TRP channels in PBMCs in IBD, the evaluation of TRP channel protein levels (enzyme-linked immunosorbent assay or immunocytochemistry), as well as modulation experiments (specific activation/inhibition, knockdown/knockout in vitro, and/or in animal models) are required. Fourth, the changes in expression levels and correlation strengths observed were very small, hence, careful attention should be paid in interpreting the data. Fifth, correlations between each TRP channel and laboratory parameters did not clearly implicate functional relationships, particularly in TRPV3, TRPV4, and TRPM5 in UC, therefore, follow up studies are needed. Finally, this study analyzed leukocytes obtained from the peripheral circulation, and not from the diseased intestine. A comparison of gene expression at these two sites could help advance our understanding of the pathophysiology of IBD.

The present results indicate, for the first time, that PBMCs from patients with IBD express di fferent mRNA levels of TRP channel members, which may play an important role in the progression of IBD. Furthermore, their expression levels in PBMCs are a promising marker for IBD. Further studies are needed to determine the clinical and pathogenic role of TRP channels in IBD.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/8/2643/s1, Figure S1: Comparison of TRP channel expression according to the current use of medications in patients with (a) ulcerative colitis (UC) and (b) Crohn's disease (CD) (b). Some of the patients received more than one category of treatment. Bars represent the median. 5ASA: 5-aminosalicylic acid; PSL: Prednisolone; IM: Immunomodulator; TNF: Tumor necrosis factor.

**Author Contributions:** K.M. designed the research; T.M., K.M., H.Y., A.M., T.Y., T.A., M.M., K.T., S.Y. (Sayo Yamasaki), K.K., S.Y. (Shinichiro Yoshioka), and H.T. performed the study and analyzed the data; K.M. and T.T. supervised the project; T.M. and K.M. co-wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported partly by a Grant-in-Aid from the Ministry of Science and Education and by Health and Labour Sciences Research Grants for research on intractable diseases from the Ministry of Health, Labour, and Welfare of Japan.

**Conflicts of Interest:** The authors declare no conflict of interest.
